The loss of OPA1 accelerates intervertebral disc degeneration and osteoarthritis in aged mice

NP cells of the intervertebral disc and articular chondrocytes reside in avascular and hypoxic tissue niches. As a consequence of these environmental constraints the cells are primarily glycolytic in nature and were long thought to have a minimal reliance on mitochondrial function. Recent studies have challenged this long-held view and highlighted the increasingly important role of mitochondria in the physiology of these tissues. However, the foundational understanding of mechanisms governing mitochondrial dynamics and function in these tissues is lacking. We investigated the role of mitochondrial fusion protein OPA1 in maintaining the spine and knee joint health in mice. OPA1 knockdown in NP cells altered mitochondrial size and cristae shape and increased the oxygen consumption rate without affecting ATP synthesis. OPA1 governed the morphology of multiple organelles, including peroxisomes, early endosomes and cis-Golgi and its loss resulted in the dysregulation of NP cell autophagy. Metabolic profiling and 13C-flux analyses revealed TCA cycle anaplerosis and altered metabolism in OPA1-deficient NP cells. Noteworthy, Opa1AcanCreERT2 mice with Opa1 deletion in disc and cartilage showed age-dependent disc degeneration, osteoarthritis, and vertebral osteopenia. Our findings underscore that OPA1 regulation of mitochondrial dynamics and multi-organelle interactions is critical in preserving metabolic homeostasis of disc and cartilage.


INTRODUCTION
Chronic low back pain, associated with intervertebral disc (IVD) degeneration, and knee and hip joint pain, a common sequela of osteoarthritis, are the leading causes of disability in the aging population 1 .In a healthy state, these tissues provide the joint with exibility and the ability to absorb applied forces.
However, with the onset of degenerative disease, these properties are lost, and the balance between cell survival, autophagy, and apoptosis becomes dysregulated leading to altered extracellular matrix (ECM) production, and increased tissue catabolism 2,3 .Nucleus pulposus (NP) cells of the IVD and articular chondrocytes reside in an avascular, nutrient-limited hypoxic environment that is hyperosmotic and express the transcription factor HIF-1α and exhibit limited replication and regenerative capacity 4,5 .
Consequently, these primarily glycolytic cells were thought to rely minimally on mitochondria to meet their energetic and biosynthetic demands [5][6][7] .However, r mounting evidence suggests that mitochondrial dysfunction promotes osteoarthritis development 3 .The deletion of mitochondrial superoxide dismutase 2 exacerbated, whereas overexpression of catalase or peroxiredoxin 3 reduced the severity of ageassociated osteoarthritis in mice, implying that mitochondrial ROS generation plays a role in the pathogenesis of osteoarthritis [8][9][10] .Moreover, we have recently showed that functional mitochondrial networks exist in NP cells (7), and to meet metabolic requirements, NP cells evidence an active mitophagic ux governed by the HIF-1α-BNIP3 axis 6 .Notably, loss of BNIP3 in NP cells resulted in mitochondrial dysfunction, affecting cellular bioenergetics, triggering meta-in ammation, and causing early onset of IVD degeneration 5 corroborating earlier studies suggesting association of mitochondrial dysfunction with disc degeneration 11 .
As dynamic organelles, mitochondria undergo ssion and fusion to control their mass and numbers.This equilibrium is governed by the ssion protein DRP1 and the fusion proteins MFN1, MFN2, and OPA1 12 .The fusion of two adjacent mitochondria promotes membrane tubulation and elongation and is a twostep process: outer mitochondrial membrane (OMM) fusion mediated by MFN1 and MFN2 followed by fusion of the inner membrane (IMM) mediated by OPA1 13 .During fusion, mitochondria control stress and energy needs by incorporating components of the damaged organelles (including mtDNA and respiration complexes) to modulate membrane potential, apoptosis, and calcium signaling 14 .The cristae of the IMM are the site of the respiratory chain complexes concerned with ATP synthesis and electron transport 15 .Notably, OPA1 regulates cristae remodeling and apoptosis, independent of mitochondria fusion 16 .Its ablation results in cristae disorganization and release of apoptotic cytochrome c 17 .
Conversely, OPA1 overexpression facilitates the formation and stability of respiratory chain supercomplexes 18 .OPA1 also functions as a sensor of metabolic changes through interactions with IMM solute transporters, facilitating the adaptation of cristae shape and respiration to cellular energy needs.Despite, the increasingly important role of mitochondria in disc and cartilage the foundational understanding of mechanisms governing mitochondrial dynamics and function in these tissues is lacking.
Herein we investigated the role of OPA1 on IVD and cartilage health.We demonstrate that loss of OPA1 in NP cells results in altered mitochondrial and cristae morphology and mass, with compromised autophagy and metabolism.For the rst time, we report the importance of OPA1 in maintaining the morphology of several organelles in the NP cells and show that conditional deletion of OPA1 (Opa1 AcanCreERT2 ) causes disc degeneration, osteoarthritis and osteopenia in aged mice.Overall, these ndings establish that the OPA1 is a critical factor required for maintaining NP and chondrocyte function and promoting the health and function of the IVD and knee articular cartilage.

OPA1 maintains mitochondrial and multi-organelle morphology in NP cells
We investigated the contribution of OPA1 in maintaining mitochondrial morphology and mass by knocking down Opa1 in primary NP cells using lentivirally delivered ShRNAs (Fig. 1A, A').OPA1-de cient NP cells showed increased mitochondrial fragmentation, evident from the smaller aspect ratio and form factor (Fig. 1A, A").TEM imaging showed that mitochondria in knockdown NP cells were smaller in size, with absent or aberrant cristae.Although some cristae were often detached, the tubular structure was preserved with narrow cristae width (Fig. 1B).Notably, OPA1-de cient NP cells contained more mitochondria but without changes in mtDNA content, indicating that the increase was due to higher ssion and not increased biogenesis (Fig. 1C, D).Moreover, the levels of the outer membrane fusion proteins MFN1, and MFN2 (Fig. S1 A, A') and the level of the ssion protein DRP1 and its receptors FIS1 and MFF were unaffected in ShOpa1 transduced cells (Fig. S1 A, A').Together these ndings showed that OPA1 de ciency affects mitochondrial number, shape, and cristae morphology without compensatory changes in other fusion and/or ssion proteins.
We determined the impact of OPA1 de ciency on the morphology of other organelles considering mitochondrial interactions with many organelles.Peroxisomes are vital for detoxi cation and ß-oxidation of very long-chain fatty acids, which subsequently transports medium-chain fatty acids to mitochondria for further breakdown.Unlike peroxisomes (PMP70 positive) in control cells with lengths between 0.1 to 1 µm, the ShOpa1 transduced cells containedhyper-tubulated and hyper-branched peroxisomes that ranged in size from 0.1 to 6 µm (Fig. 1E, E').We also examined the morphology of EEA1 positive early endosomes, which receive cargo and sort it into recycling and degradative compartments.OPA1-de cient NP cells exhibited enlarged endosomes ranging in size from 0.5-2 µm, as opposed to endosomes that were smaller than 0.7 µm in control cells (Fig. 1E, E").We also investigated the morphology of the Golgi complex as the primary secretory pathway organelle.There was a pronounced fragmentation of cis-Golgi (GM130 positive) in ShOpa1 transduced cells (Fig. 1E, E"', Fig. S2A).In contrast, TGN46-stained trans-Golgi, as well as RAB7-stained late endosomes, and LAMP1-stained lysosomes were unaffected (Fig. S2B).These ndings suggest that in addition to maintaining mitochondrial and cristae morphology, OPA1 is essential for sustaining the morphology of organelles that include peroxisomes, early endosomes, and the cis-Golgi in NP cells.

OPA1 deletion disrupts NP cell autophagy
A striking observation was that OPA1-de cient NP cells showed diminished LC3B-positive autophagosomes (Fig. 2A) while Western blot analysis showed signi cantly lower LC3II and p62 levels in ShOpa1 cells.These ndings suggested an overall reduction in autophagy (Fig. 3B, B').Since we have recently shown that BNIP3 activation and mitochondrial translocation are required for hypoxia-induced mitophagy in NP cells 6,19 , we investigated whether BNIP3 mitochondrial localization is affected by OPA1 knockdown.Indeed, we observed BNIP3 sequestration in the nucleus of OPA1-de cient cells, without alteration in NIX localization (Fig. S1B, B') and protein levels (Fig. S1C, C').The levels of other autophagyrelated proteins, BECLIN1, and the ATG12-ATG5 complex remained unaffected whereas those of the apoptotic transcription factor CHOP decreased suggesting ShOpa1 cells did not activate apoptosis (Fig. S1C, C').Moreover, levels of ubiquitinated proteins (Fig. S1D, D'), PARK2, and its substrate phosphoubiquitin, were unaffected by OPA1 knockdown (Fig. 2B, B', C, C') 20 .To ascertain whether OPA1 deletion impacts autophagy initiation and/or rate of degradation, we treated cells with ba lomycin A 1 for 2 hours and noted failure to enhance LC3II accumulation in the ShOpa1 cells (Fig. 2D, D').These data indicate dysfunction in the autophagic pathway of OPA1-de cient NP cells.

OPA1-de cient NP cells show dysregulated bioenergetics
Since OPA1-de cient NP cells showed mitochondrial fragmentation and abnormal cristae, we measured their ATP production rates from glycolysis and oxidative metabolism by integrating ECAR and OCR measurements (Fig. 3A, B) 5,21 .Under basal conditions, OCR was signi cantly higher, while ECAR remained unchanged, suggesting OPA1-de cient NP cells consumed more oxygen (Fig. 3A, B, C).Following the addition of glucose, ShOpa1 transduced cells showed similar ECAR pro les to ShCtrl group, but the OCR remained signi cantly higher and similar to the levels observed under basal conditions (Fig. 3C).Further determination of glycolytic ATP generation showed a signi cant increase in ShOpa1 cells; however, oxidative ATP production was comparable between groups despite higher oxygen consumption by OPA1-de cient NP cells.These data suggest uncoupling of OXPHOS in OPA1-de cient NP cells, in conjunction with the aberrant cristae morphology.We also investigated the effect of OPA1de ciency on the glycolytic capacity of NP cells 5,22 by measuring ECAR and OCR pro les under basal (no substrate) conditions and following sequential addition of glucose, rotenone + myxothiazol, and monensin + FCCP (Fig. 3E, F).The ShOpa1 cells showed no difference in EACR but demonstrated a prominent increase in OCR (Fig. 3E, F).However, the basal glycolytic rate, maximum glycolytic capacity, ATP demand-limited rate, and glycolytic reserve computed from H+/lactate production were unaffected (Fig. 3G).Overall, these results suggest that OPA1-de ciency compromises energy metabolism of NP cells.

OPA1-de cient NP cells evidence altered glucose and glutamine metabolism
To understand the broader metabolic implications of OPA1 de ciency, we performed widely targeted metabolic pro ling on NP cells.A total of 261 metabolites that satis ed the QC limit of < 30% coe cient of variation (CV) were imported individually into the SIMCA-p program for multivariate analysis.An unsupervised principal component analysis (PCA) and supervised partial least square-discrimination analysis (PLS-DA) models were established which showed a clear separation between the ShOpa1 and ShCtrl samples (Fig. 4A, B).We identi ed 36 downregulated and 9 upregulated metabolites (FDR ≤ 0.05) in OPA1-de cient cells (Fig. 4C).Concerning altered metabolites, the levels of glycolytic metaboliteglucose-6-phosphate and TCA metabolite malate, as well as the collagen-related amino acids hydroxyproline, and hydroxylysine were raised (Fig. 4D-G), whereas fatty acid metabolites stearic acid and acetylcarnitine and the one carbon metabolites s-adenosyl homocysteine and s-adenosyl methionine were signi cantly lower in OPA1 knockdown cells.(Fig. 4H-K).Additionally, the ShOpa1 group showed a signi cant increase in NADP with lower AMP, without affecting the overall ADP and ATP levels (Fig. 4L-O).However, AMP/ATP and ADP/ATP ratios were affected in knockdown NP cells (Fig. 4P, 4Q).Analysis of upregulated metabolites revealed enrichment in galactose metabolism, nucleotide sugar metabolism, starch, and sucrose metabolism, ascorbate and aldarate metabolism, pentose and glucuronate interconversion, sphingolipid metabolism, pentose phosphate pathway activity, glycolysis/gluconeogenesis, and inositol phosphate metabolism-related metabolites.Similarly, metabolic pathway analysis of upregulated entities showed nucleotide sugar metabolism, malate-aspartate shuttle activity, transfer of acetyl group into mitochondria, and starch and sucrose metabolism, and lactose synthesis were the most impacted pathways in ShOpa1 transduced cells (Fig. 4R, Fig. S3A).Arginine, cysteine, methionine, proline, alanine, aspartate, and glutamate were among the enriched metabolites that were signi cantly downregulated, and the enrichment analysis showed downregulation of aminoacyl-tRNA biosynthesis, pantothenate and CoA biosynthesis, pyrimidine and purine metabolism and taurine and hypotaurine metabolism in ShOpa1 cells.Likewise, the most impacted downregulated pathways in ShOpa1 cells were spermidine and spermine biosynthesis, and betaine, urea, aspartate, methionine, arginine, and protein, taurine and hypotaurine, pyrimidine, and pantothenate and CoA metabolism, and ammonia recycling (Fig. 4S, Fig. S3B).
To further delineate the utilization of major metabolic substrates glucose and glutamine, OPA1-silenced NP cells were cultured for 24 hours under hypoxia with a 50% enrichment in either [1,2]-13 C-glucose or U 13 C-glutamine (Fig. 5A).Based on the lactate and glutamate of the medium we calculated the glycolytic, pentose cycle, PDH, PC, PDH + PC, and PHD/PC ux, as previously described 5 .OPA1-silencing did not affect glucose ux through glycolysis or the pentose cycle (Fig. 5B, C). 13 C enrichment in medium glutamate provided a measure of glucose ux into the TCA cycle via PDH and PC.PDH ux was signi cantly decreased, without altering ux through PC; combined PDH + PC ux or the PDH/PC also remained unchanged (Fig. 5D-G).
Analysis of extracted metabolites from the cell pellet showed a modest increase in sigma mean (∑ mn = 1 *M1 + 2*M2 + 3*M3, etc.) of alanine (m/z 260) and a decrease in serine (m/z 390) with minimal changes in lactate (m/z 261), citrate (m/z 591), and succinate (m/z 289).A small decrease in M1 glutamate (m/z 432) with minimal change in palmitate (m/z 313) as well as enhanced enrichment in stearic acid (m/z 341) was also noted (Fig. 5H-O).When U 13 C-glutamine utilization was measured, ShOpa1 cells evidenced a reduction in TCA cycle intermediates citrate (m/z 591), succinate (m/z 289), fumarate (m/z 287), malate (m/z 419), and aspartate (m/z 418), without changes in the enrichment of lactate (m/z 261) (Fig. 5P-U).Both hypoxia and ETC inhibition are shown to increase the levels of succinate 23 .Interestingly, however, our 13 C-MFA data showed increased succinate ∑ mn compared to fumarate in both ShCtrl and ShOpa1 cells, suggesting impairment of the oxidative TCA cycle.Furthermore, when we assessed succinate oxidation (fumarate M + 4/succinate M + 4) and fumarate reduction (succinate M + 3/fumarate M + 3), no change in the oxidation, with a downward trend in fumarate reduction was noted (Fig. 5V, W).These studies lent support to the notion that OPA1 is required for optimal NP cell metabolism.

Conditional deletion of Opa1 in IVD accelerates age-associated degeneration
To gain a better understanding of how OPA1 impacts spinal health, we generated Opa1 conditional knockout mice by administering tamoxifen to 3-month-old Acan CreERT2 Opa1 / (Opa1cKO) and Opa1 / (WT) mice (Fig. 6A, B) 24 .In adult mice, the Acan-Cre ERT2 driver is highly effective in targeting all three compartments of the IVD as well as the articular and growth plate cartilages 25 .The successful deletion of OPA1 in IVD was con rmed by mRNA and protein evaluation of the NP and annulus brosus (AF) tissues (Fig. 6C, D).We performed a quantitative histopathological analysis of IVD morphology using the Modi ed Thompson grading on 7, and 12-month-old Opa1cKO mice (Fig. S4) which indicated no noticeable degeneration in the IVD in the lumbar (Fig. S4.A-A", B-B") or caudal regions of the spine (Fig. S4.C-C", D-D").At 12 months, however, caudal discs of Opa1cKO mice showed changes in NP cell morphology and AF hyperplasia (Fig. S4D"').When Opa1cKO mice were evaluated at 20 months, a prominent degenerative phenotype in the NP and AF tissues of caudal IVDs compared to WT mice was evident (Fig. 6E).The distribution of Modi ed Thompson grading scores showed a signi cantly higher proportion of Opa1cKO discs had NP and AF compartments scores of 3 or 4, indicating severe degeneration (Fig. 6E', E").At 20 months, the degenerative phenotype was characterized by a diminished SafraninO stained NP extracellular matrix, a signi cant loss of NP cells with the remainder of cells acquiring a hypertrophic chondrocyte-like morphology and irregularities in AF lamellar organization (Fig. 6E).There was also a clear loss of demarcation between NP and AF tissue boundaries in Opa1cKO mice (Fig. 6E).Those discs that retained notochordal NP cell bands displayed morphological changes such as loss of cytosolic vacuoles (Fig. 6E"').In addition to these morphological changes, Opa1cKO mice exhibited AF hyperplasia re ected in increased tissue area (Fig. 6F, F').
Picrosirius red staining coupled with polarized light imaging was used to ascertain alterations in collagen matrix organization (Fig. 6G).At 20 months, Opa1cKO mice showed the presence of collagen bers in the NP compartment.Moreover, when the fraction of brous tissue area in the NP was measured; Opa1cKO animals also exhibited a higher proportion of brous tissue area than a few discs in WT mice with NP brosis (Fig. 6G').In the AF tissue, Opa1cKO mice showed a higher proportion of thin collagen bers suggesting increased turnover (Fig. 6G", G"').Notably, in 20-month-old mice lumbar IVDs showed a milder phenotype compared to caudal discs; there was a higher proportion of lumbar discs with NP and AF compartments scoring grade 4 but when scores of all discs were averaged it did not reach statistical signi cance (Fig. S5A, A', A").Similar to caudal IVDs, the NP compartment of lumbar IVDs in 20-monthold Opa1cKO mice displayed a higher prevalence of brous tissue underscoring degeneration (Fig. S5B,  B').

OPA1 deletion affects NP cell phenotype and alters the IVD matrix composition
We assessed the NP cell phenotype in 20-month-old mice by measuring the abundance of the phenotypic markers, carbonic anhydrase (CA3), and glucose transporter I (GLUT1).Strikingly, expression of these markers was lost by the few cells that persisted in the NP compartment, suggesting the native notochord cell population underwent a phenotypic switch (Fig. 7A, B).
Considering the NP cells may have undergone a phenotypic shift, we stained IVD sections for COLX, a marker of hypertrophic chondrocytes.Staining was considerably higher in both the NP and AF of Opa1cKO mice (Fig. 7C), suggesting that resident cells in the NP compartment acquired hypertrophic chondrocyte-like characters.We also noted that Opa1cKO had a lower AF abundance of collagen I (COLI) (Fig. 7D, D') and cartilage-oligomeric matrix protein (COMP), an important non-collagenous matrix component (Fig. 7D, D").While ACAN staining revealed no major differences across genotypes (Fig. 7E,   E'), the cells in the NP and AF compartments of of Opa1cKO showed a robust increase in the pericellular staining of ARGxx, an ACAN neoepitope formed by ADAMTS4/5 dependent cleavage (Fig. 7E, E").Overall, these ndings suggest that OPA1 is required for maintaining the IVD cell phenotype and major structural components of the ECM.

Opa1 cKO mice evidence alterations in vertebral bone health
To investigate the impact of OPA1 deletion on vertebral bone health we performed micro-computed tomography (µCT) on the lumbar (L3-6) vertebrae of Opa1cKO and WT mice.In WT mice, threedimensional (3D) reconstructions showed expected trabecular bone loss with aging; however, Opa1cKO mice exhibited a signi cant reduction in vertebral trabecular bone architecture at 7-and 12-months (Fig. 8A).Thus, bone volume/trabecular volume (BV/TV), bone mineral density (BMD), trabecular thickness (Tb.Th), and trabecular number (Tb.N) was decreased.The change in bone structure in the 7 and 12-month-old Opa1cKO was maintained at 20 months implying early onset osteopenia (Fig. 8B-E).As expected, trabecular separation was also greater in 7-month-old Opa1cKO mice (Fig. 8F) and the structural model index (SMI), a parameter that identi es the rod-like structure of trabeculae and is linked to bone strength and fracture risk, was considerably greater at 7 and 12 months (Fig. 8G).Interestingly, unlike early changes noted in trabecular bone of Opa1cKO mice, changes in cortical bone structure were evident predominantly at 20-months, with a substantial increase in mean total cross-sectional bone area (B.Ar.), tissue area (T.Ar.) and the cross-sectional thickness (Cs.Th.), without a change in tissue mineral density (TMD) (Fig. 8H-L).We also noted an increased vertebral length and disc height index (DHI) in Opa1cKO mice at 7-and 12-months (Fig. 8M-P), which has been correlated with IVD degeneration 26 .These ndings suggest that OPA1 plays an important role in the control of vertebral bone health.
Opa1 cKO mice show increased severity of age-associated OA Since the Acan CreERT2 allele e ciently targets articular cartilage, we investigated if OPA1 deletion has deleterious effects on knee articular cartilage and overall knee joint health in mice.µCT images showed evidence of bone spurs in the knee joints of 20-month-old but not 12-month-old Opa1cKO mice (Fig. 9A, Fig. S6A).µCT was also used to evaluate tibial subchondral bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and subchondral bone plate thickness (SCBP) in the medial and lateral tibial plateaus.These analyses showed a signi cant increase in subchondral bone thickness in the lateral compartment of the 20-month-old mice (Fig. 9B-E); none of the measured bone metrics showed changes at 12 months (Fig. S6B-E).To study cartilage structure, we stained 12-and 20month-old knee joints sectioned in the mid-coronal plane with H&E and toluidine blue as we have previously described 27 (Fig. 9F, G).At 20 months, Opa1cKO mice showed severe OA with loss of articular cartilage on the lateral tibial plateaus and femoral condyle, but only minor degradation of articular cartilage in the medial compartment (Fig. 9F).Furthermore, H&E staining revealed osteophyte formation in the medial compartment of WT mice, and osteophyte formation in both the lateral and medial compartments of Opa1cKO animals (Fig. 9G).When these structural changes were quanti ed, Opa1cKO mice exhibited a signi cant increase in articular cartilage score (ACS) in the lateral knee compartment at 20-months.However, there were no deviations in toluidine blue scores and osteophyte scores between knee compartments or genotypes (Fig. 9H-J).However, the cumulative osteophyte score in Opa1cKO mice was signi cantly higher than in WT (Fig. 9K).Furthermore, H&E staining showed synovial hyperplasia/ossi cation in the lateral and medial knee compartments of 20-month-old Opa1cKO mice (Fig. 10A) however, there were no morphological changes between WT and Opa1cKO mice at 12 months (Fig. S6.F, G).Additionally, we conducted histomorphometric analysis on both lateral and medial knee joint compartments of 12 and 20-month-old mice.In the lateral compartment of 20-month Opa1cKO mice there was a signi cant decrease in the area and thickness of articular cartilage and calci ed cartilage (Fig. 10B, B', C, C', D, D').SCBP area and thickness were signi cantly increased in Opa1cKO mice when compared to controls, indicating enhanced subchondral bone sclerosis in OPA1 loss mice, a nding that aligns with our µCT data (Fig. 10D, D').We also noted a considerable increase in the synovial hyperplasia/ossi cation score in the lateral joint compartment of 20-month-old Opa1cKO mice (Fig. 10E).On the other hand, 12-month-old mice showed no signi cant morphological and histomorphometric changes or evidence of an OA phenotype (Fig. S6F-M').It is interesting to note that the profound OA phenotype in Opa1cKO mice was primarily observed in the lateral compartment as opposed to the medial compartment which is typically more affected.These ndings demonstrate that cartilage-speci c loss of OPA1 enhances the severity of age-associated OA in mice.

DISCUSSION
This study for the rst time establishes a causal relationship between sustaining mitochondrial dynamics through OPA1 and maintenance of the spine and knee joint health during aging in mice.The reliance on mitochondrial function was notable since hypoxic chondrocytes and in particular NP cells of IVD primarily rely on glycolysis for energy production 5,6 .Relevant to this nding, we have previously shown the existence of mitochondrial networks in NP cells (7) and that mitochondrial activity is governed by the HIF-1α-BNIP3 axis controlling mitophagic ux 6 .We also demonstrated that deletion of mitophagy receptor BNIP3 in NP cells caused mitochondrial dysfunction affecting bioenergetics, triggering metain ammation, and resulting in early disc degeneration in mice 5 .Moreover, our transcriptomic analyses of human NP tissues showed an association of biological themes related to mitochondrial functions with disc degeneration 11 .Herein, we show that, OPA1 controls the morphology of mitochondria and cristae as well as multiple organelles including peroxisomes, early endosomes, and cis-Golgi, and that OPA1-loss results in dysregulated autophagy in NP cells.Further, we demonstrate that Opa1 AcanCreERT2 mice evidence accelerated age-dependent IVD degeneration, vertebral osteopenia, and severe OA of knee joints.Our ndings highlight the fact that dysregulation of mitochondrial dynamics affects the metabolism, organelle integrity, and the autophagic/mitophagic pathway causing disc and cartilage degeneration in mice.
OPA1 is required for IMM fusion as well as the maintenance of cristae shape and its mutations are linked to multiple pathologies including vision impairment 28 , developmental delay, muscle-related disorders, peripheral neuropathy, and cardiomyopathy 29 .Concerning axial skeleton, we have previously showed that changes in mitochondrial morphology impact NP cell metabolism and mitophagy and pathways related to mitochondrial dysfunction are enriched in transcriptomes of degenerated human NP tissues 5,6 .Herein we observed that deletion of OPA1 resulted in a reduction in mitochondria size but an increase in numbers.This change was unlikely due to mitochondrial biogenesis since there was no difference in the mtDNA content of the mutated cells. 30,31.More than likely, as the OPA1-de cient NP cells lacked cristae or exhibited detached cristae it indicated that OPA1 was required for IMM invagination and cristae development.Moreover, it is known that mitochondria interact with other organelles and maintain cellular homeostasis while maintaining mitochondrial function 32 .From this perspective, impact on peroxisomes, endosomes, and cis-Golgi morphology suggested that OPA1 is critical for preserving multi-organelle morphology likely through governing their interactions with mitochondria in NP cells.To the best of our knowledge, this is the rst report on the involvement of OPA1 in sustaining the morphology of these organelles.
When mitochondrial quality control was assessed, a signi cant decrease in key autophagy-related proteins p62 and LC3-II in OPA1-knockdown cells was noted.Moreover, Ba lomycin A 1 treatment showed a lack of LC3-II accumulation suggesting impaired autophagosome formation.LC3-II targets ubiquitinpositive cargo to sequester them into developing autophagosomes 33 .Notably, these autophagy proteins are subjected to regulatory post-translational phosphorylation by mTORC1 (negative) and ULK1 and AMPK (positive) whereby AMPK promotes autophagy by phosphorylating ULK1 when the ADP/ATP or AMP/ATP ratio is elevated.Indeed, our metabolic pro ling indicated that decreased AMP/ATP and ADP/ATP ratios would be associated with decreased phosphorylation, and, as a consequence, reduced autophagy.In contrast to previous studies demonstrating enhanced mitophagy in OPA1 defective cells 31,34,35 , we noted that OPA1-knockdown not only inhibited selective autophagy (mitophagy) but also macroautophagy, an event that in uenced the morphology of multiple organelles, including the ER, endosomes, and Golgi 36-38 .For example, enlarged early endosomes and fragmented Golgi, together with autophagosome accumulation is implicated in neurodegenerative diseases such as Down syndrome and Parkinson's, 39,40 .Importantly, changes in these organelles underscore the defects in autophagy and disruptions in endocytic and secretory pathways in OPA1-de cient NP cells.Together, our data supports the hypothesis that OPA1 regulates organelle morphology and autophagic/endocytic/secretory pathways in NP cells.One important outcome of these ndings is that autophagy and mitochondrial dysfunction are associated with aging, disc degeneration, and the pathogenesis of OA 2,41,42 .
Considering the relationship between OPA1 and mitochondrial function, it is known that in many cell types, which primarily rely on oxidative ATP generation, structural changes in mitochondria profoundly alter energy metabolism.However, since NP cells are predominantly glycolytic, it was important to determine whether alterations in mitochondrial shape and cristae morphology in uenced NP cell bioenergetics.We found that while fragmented mitochondria with aberrant cristae morphology consumed more oxygen, it did not manifest in increased oxidative ATP production rates.The number of ATP molecules generated for each dioxygen molecule consumed might vary depending on the mitochondrial e cacy 43,44 , suggesting mitochondrial dysfunction in the ShOpa1 transduced NP cells.Our ndings also differed from prior work showing lower OCR and oxidative ATP generation in OPA1-de cient cells 45,46 but were in agreement with the increase in glycolytic ATP production rate noted in OPA1-de cient neutrophils 47 .Overall, the results of the current study underscored the observation that OPA1 de ciency in uences the energy metabolism of NP cells.
In addition to bioenergetics, the mitochondrion is also the primary site for biosynthetic pathways.Metabolic systems in OPA1-de cient NP cells that were most negatively impacted involved spermidine and spermine biosynthesis and betaine metabolism.Likewise, there was a decrease in amino acid metabolism, taurine and hypotaurine synthesis, pyrimidine, pantothenate and CoA biosynthesis, and urea cycle metabolite.In agreement with these ndings, OPA1 deletion in MEF cells resulted in decreased levels of metabolites associated with spermidine and spermine and taurine and hypotaurine pathways 48 .
Notably, spermidine and taurine levels are shown to decline with age, and mitochondrial dysfunction is one of the primary contributors to their de ciency 49,50 .Supplementation of spermidine and taurine has been proven to prolong longevity in mice via boosting autophagy/mitophagy, mitochondrial biogenesis, and mitochondrial respiration 49,50 .Interestingly, the increased levels of glucose-6-phosphate was, not attributable to enhanced glycolysis associated with the lack of corresponding increase in intracellular and extracellular lactate levels.Depending on cellular metabolism, glucose-6-phosphate shuttles between glycolysis, pentose phosphate shunt, hexosamine biosynthetic pathways, gluconeogenesis, and de-novo lipid synthesis 51 .Accordingly, in ShOpa1 cells, glucose-6-phosphate was utilized for the synthesis of NADP, nucleotide sugar metabolism, and gluconeogenesis pathway activity.Furthermore, elevated malate and the increased malate-aspartate shuttle activity are linked to gluconeogenesis, while increased serine levels in ShOpa1 cells indicated de novo serine synthesis, implying that malate was exported to the cytosol 52,53 .Interestingly, acetylcarnitine level was decreased in OPA1-de cient NP cells, which links mitochondrial metabolism to histone acetylation and lipogenesis 54 .In agreement with a previous report showing OPA1 regulation of lipid metabolism, we also observed reduced fatty acid metabolism 55 .OPA1 de ciency also affected one-carbon metabolism with increased serine biosynthesis but decreased utilization in ShOpa1 cells.This reduction was likely due to a decrease of s-adenosyl homocysteine and s-adenosyl methionine co-substrates, which are involved in transferring methyl groups for the synthesis of DNA, amino acids, and polyamines.This was further supported by reduced pyrimidine metabolism, polyamines, and amino acids metabolism.Notably, serine biosynthesis and one-carbon metabolism are linked to a variety of mitochondrial disorders 56 .In summary, the metabolite pro ling studies revealed broad metabolic dysregulation in OPA1-de cient NP cells. 13C isotope labeling experiments utilizing two stable isotope tracers [1,2]-13 C-glucose and U 13 C-glutamine shed further insights into metabolic dysregulation of OPA1-de cient NP cells.The use of [1,2]-13 C-glucose MFA revealed a reduction in PDH ux despite an increase in alanine ∑ mn indicating an elevated pyruvate pool.We also noted a decrease in intracellular M + 1 glutamate enrichment, an indirect measurement for alpha-ketoglutarate, suggesting pyruvate is prevented from entering the TCA cycle via the conventional pathway.Furthermore, since there was no change in PC ux, this supports the anaplerosis i.e. pyruvate carboxylation replenishes TCA intermediates and allows the Krebs cycle to continue since the intermediates are not only important for macromolecule synthesis but also for protein post-translational modi cations, chromatin modi cation, and DNA methylation 57 .Interestingly, in solid hypoxic tumors, increased anaplerosis through PC is required for extracellular collagen production and aberrant brosis by tumor-associated broblasts, a phenotype we have noted in the discs of Opa1cKO mice 58 .Regarding glutamine utilization, the labeled glutamine entered the TCA via an anaplerotic reaction to alpha-ketoglutarate with M + 4 labeling of the forward TCA cycle intermediates succinate, fumarate, malate, and citrate.In contrast to glucose, glutamine labeling showed decreased TCA cycle intermediates such as citrate, succinate, fumarate, malate, and oxaloacetate (aspartate) enrichment.The Citrate M + 4 label signi es the oxidative (forward) TCA cycle while the M + 5 label is for the reductive (reverse) TCA cycle.We noted a substantial reduction in citrate M + 5 label (~ 2.5:1 M + 4:M + 5 citrate), indicating a modest reversal in TCA ux.Of interest, under normal physiological conditions, the majority of succinate is generated by oxidative or forward TCA cycle.Recent studies found that under hypoxic conditions a part of succinate was generated from fumarate or when ETC was inhibited, implying that fumarate reduction to succinate may act as a valve for surplus electrons from the ETC.Further, it was reported that fumarate was catalyzed explicitly by complex II, rather than passively collecting leaky electrons from the ETC 23 .Interestingly, our 13 C MFA data showed increased succinate ∑ mn compared to fumarate in both ShCtrl and ShOpa1 cells, suggesting an oxidative TCA cycle.Furthermore, when we assessed succinate oxidation (fumarate M + 4/succinate M + 4) and fumarate reduction (succinate M + 3/fumarate M + 3), we observed no changes in the oxidative process.Overall, these ndings suggested that NP cells do not experience metabolic stress by their physiologically hypoxic niche.These studies suggested that mitochondrial morphology and cristae architecture are not only central to energy metabolism, but the integrity of these structures is critical for the proper functioning of mitochondria in NP cells.
Finally, we investigated the in vivo function of OPA1 in the IVD and knee cartilage.We found that Opa1cKO mice evidenced enhanced IVD degeneration with aging.Interestingly, the caudal discs showed a more pronounced phenotype than the lumbar discs.Previous studies have shown that caudal discs of mice are more prone to metabolic dysregulation and subsequent degeneration with aging 59 .Moreover, the caudal spine experiences relatively lower axial loading and different motions than the lumbar spine, it is therefore not unreasonable to hypothesize that the unique interactions between genetics and environmental factors produce varying phenotypic outcomes across different spine regions 60 .Similar to IVD degeneration, aged Opa1cKO displayed enhanced age-associated OA severity; the disease state was characterized by enhanced cartilage damage, osteophyte formation, subchondral bone sclerosis and synovial hyperplasia and/or ossi cation.While the medial compartment of the knee is usually prone to cartilage degeneration 61 , interestingly, the lateral knee compartment in Opa1cKO mice exhibited signi cantly more pronounced OA.Similar to caudal discs, the lateral knee compartment experiences lower compressive loads 62 .Moreover, consistent with previous studies that demonstrate exercise and/or loading in human and animal models can partially restore the functionality of faulty mitochondria 63,64 , lack of substantial degeneration in lumbar discs and the medial compartment of the knee suggests a protective effect of loading on these joint tissues 60,65 .Regarding the age-dependency of the phenotypes, of course, in hypoxic skeletal tissues, chronic metabolic stress arising from abnormalities in mitochondrial function would be expected to in uence tissue function, especially in aging individuals.
It is important to note that similar to the NP and knee cartilage, signi cant degenerative changes affect the AF in Opa1cKO mice, underscoring the important contribution of mitochondrial activity to annulus tissue health.This observation is supported by a previously reported microarray analysis of human AF tissues that showed altered genes related to mitochondrial function during degeneration 66 .Moreover, similar to OA development, in degenerated AF tissues, ROS-related gene expression is dramatically altered, suggesting that mitochondrial dysfunction and ROS production promote AF degeneration 66 .
These in vivo ndings coupled with our mechanistic studies suggest that the mouse IVD, spinal column, and joint cartilage phenotypes is the outcome of metabolic dysregulation and cumulative degenerative processes driven by OPA1 deletion.Moreover, in many soft tissues, age-related pathologies, and metabolic disorders due to the accumulation of faulty mitochondria has been exhaustively demonstrated 67,68 .From this perspective, targeting and modifying the autophagic pathway and preserving mitochondrial function should be of major concern when designing therapeutics to treat diseases linked to degenerative musculoskeletal conditions.

Organelles morphology analysis
Mitochondria, peroxisomes, endosomes and Golgi number, branching and morphology were quanti ed in ImageJ using methods reported earlier 5,6 .Brie y, the confocal images were converted to binary by threshold and then converted to a skeleton that represented the features in the original image using a wireframe of lines one pixel wide.All pixels within a skeleton were then measured using analyze skeleton.
The output will give the number of particles which denotes the total number of mitochondria, the aspect ratio (AR) represents the "length to width ratio" and the form factor (FF), the complexity and branching aspect of mitochondria were calculated from circularity.For the endosome, Feret diameter was used to plot the graph.

Seahorse XF analysis
Maximum glycolytic capacity and ATP production rate using methods reported by Mookerjee and colleagues 5,21,22 .In brief, ShCtrl, ShOpa1 cells were plated in a 24-well Seahorse V7-PS test plate under hypoxia 24 hours before the experiment.Cells were washed three times with 500 µl of KRPH (Krebs Ringer Phosphate HEPES) before being cultured for one hour at 37 o C in 100% air.For glycolytic capacity calculation, oxygen consumption rate (OCR) and related extracellular acidi cation rate (ECAR) were determined in a Seahorse XFe24 analyzer (Agilent Techonoligies) by adding 10 mM glucose, 1 µM rotenone plus 1 µM myxothiazol, and 200 µM monensin plus 1 µM FCCP via ports A-C.OCR and ECAR were assessed by adding 10 mM glucose, 2 µg oligomycin, 1 µM rotenone plus 1 µM myxothiazol to determine ATP generation rate from oxidative and glycolytic pathways.
Widely targeted small metabolite measurements Cells were transduced with ShCtrl and ShOpa1 viral particles as described above.On the third day, the medium was changed to DMEM without pyruvate, 10% dialyzed FBS (Sigma F0392), and the cells were grown under hypoxia for 24 hours.Cells were washed and collected in ice-cold 80% methanol before being snap-frozen in liquid nitrogen and kept at -80 degrees Celsius until use.Prior to analyzing the metabolites, cell pellet samples were centrifuged and pipetted into a LC sampling vial.Each sample had internal standards.After drying under mild nitrogen ow, the samples were reconstituted in 150 µl of 80% methanol for injection.The samples were analyzed on an ABsciex 6500 + coupled with a Waters UPLC.Small metabolites were separated using the Ace PFP column and the iHILIC-p column (HILICON) and a pooled quality control (QC) sample was added to the sample list.The QCs sample was injected six times to calculate the coe cient of variation (CV) for data quality control.Metabolites with CVs lower than 30% used for the quanti cation.MetaboAnalyst 5.0 web server was used to analyze the data, and acceptable metabolites were manually input using the HMDB number.The small metabolites pathway data bank (SMPDB), which contains 99 compounds based on normal human metabolic pathways, was used for enrichment and pathway analysis.MetaboAnalyst provides the list of pathways in which these metabolites are found. 13

C-Metabolic ux analysis
For [1,2]- 13 C-glucose ux analysis, 50% of DMEM media contained 13 C-labeled glucose.A volume of 100 µl of cell culture medium from 1,2-13 C glucose experiment was treated with 400 µl of methanol.After centrifugation, the supernatant was transferred to a LC-MS sampling vial and dried under gentle nitrogen ow.The sample was reconstituted into 100 ml of 80% methanol for LC-MS injection.Metabolite separation was performed on an ACE PFP-C18 column (1.7 µM x 1 mm x 100 mm) and analyzed on a ABSciex 6500 + with a Multiple Reaction Monitoring (MRM) mode.The glycolysis, pentose cycle, PDH, PC, PDH/PC, and PDH + PC uxes were calculated using methods reported by Madhu et al., 5 .
When ux was assessed in the U 13 C-glutamine labeling experiment, labeled glutamine was added to be 50% of the total DMEM glutamine concentration.Methanol extraction from [1,2]-13 C-glucose and U 13 C glutamine labeled cell pellets were dried under gentle nitrogen ow.The dried samples were derivatized with a methyl-moximation (with 15 mg/ml methoxy amine in pyridine, 30 o C for 90 minutes) and MTBSTFA (at 70 o C for 60 minutes).The samples were then analyzed with an Agilent GC-MS, with an electron impact mode and a DB-5MS column (Agilent) following our protocol 5 .The data were analyzed with Mass Hunter Quantitative Analysis software (Agilent).The enrichment was calculated after subtraction from the background of the non-labeled treatment samples.Fractional enrichments from G to R is sigma (stands for sigma mean), and it is equal to the weighted mean average of the metabolite's enrichment (sigma mn = 1 xm1 + 2xm2 + 3xm3, etc).

Generation of conditional knockout mice
All mouse studies were carried out in conformity with the Institutional Animal Care and Use Committee (IACUC) of Thomas Jefferson University's applicable rules and regulations.Opa1 / on mixed C57BL/6-129/SvEv background were described previously 46 .Aggrecan-CreERT2 mice were from Jackson Laboratories (stock #019148).OPA1 conditional knock-out (Opa1cKO:Acan CreERT2 Opa1 / ) and control (Opa1CTR: Opa1 / ) mice were generated and analyzed after 3 (7-month-old), 9 (12-month-old), and 17 (20-month-old) months after tamoxifen injections to determine degree of degeneration.For all experiments, skeletally mature 3-month-old female and male mice of all genotypes received an intraperitoneal injection of 100 mg/kg tamoxifen (Sigma-Aldrich, St. Louis, MO, USA) dissolved in palm oil (Sigma-Aldrich) for 3 consecutive days to activate Cre recombinase.

Immunohistochemistry and confocal analysis
Mid-coronal 7 µm thick disc tissue sections were de-para nized and incubated in microwaved citrate buffer for 20 min, proteinase K for 10 min at room temperature, or Chondroitinase ABC for 30 min at 37°C for antigen retrieval.Sections were blocked in 5% normal serum (Thermo Fisher Scienti c, 10000 C) in PBS-T (0.4% Triton X-100 in PBS) and incubated with antibody against GLUT-  Mouse hindlimbs (right limb) were harvested, surrounding muscles were removed, and limbs were xed in formalin (10%, 48 hours).MicroCT analysis of the femur, tibia, and knee joint were performed on a Bruker SkyScan 1275 scanner as we have previously described 27 .MicroCT reconstructions and quantitative analysis of tibial subchondral bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and subchondral bone plate thickness (SCBP) were performed using the SkyScan CT Analyzer (CTan) and CT Vox software on coronal slices that spanned the medial and lateral tibial plateaus.
Histological and histomorphometry analysis of OA Hindlimbs were decalci ed (EDTA, 19%, 21 days), processed, para n embedded, and sectioned along the coronal plane, as we have described 27 .Mid-coronal sections were stained with hematoxylin and eosin (H&E), or toluidine blue and OA severity was analyzed by Articular Cartilage Structure (ACS), toluidine blue, and osteophyte scoring on the medial and lateral tibial plateaus (MTP, LTP) and femoral condyles (MFC, LFC).Synovial hyperplasia was assessed using a 0-3 scale as described earlier 71 In joints that presented with synovial ossi cation, a maximal synovial hyperplasia score was assigned.Analysis was performed by a blinded scorer with experience of the OA scoring techniques.Detailed histomorphometric analysis of articular cartilage thickness and area, calci ed cartilage thickness and area, and subchondral bone thickness and area were analyzed on the MTP and LTP using ImageJ software as we have previously described 27 .

Statistical analysis
Statistical analysis was performed using Prism9 (GraphPad, La Jolla, CA, USA).
Micro-computed tomography (µCT) analysesµCT imaging was performed on the lumbar spine of 7, 12, and 20-month WT and Opa1cKO mice using the high-resolution µCT scanner (Skyscan 1272, Bruker, Belgium).Lumber segments L2-6 were placed in PBS and scanned with an energy of 50 kVp and current of 200 mA resulting in 15 mm 3 voxel size resolution.Trabecular parameters were assessed in the 3D reconstructed trabecular tissue using Skyscan CT analysis (CTAn) software by contouring the region of interest (ROI).The bone volume percentage (BV/TV), trabecular number (Tb.N.), trabecular thickness (Tb.Th.), and trabecular separation (Tb.Sp.) of the resulting datasets were all evaluated.Cortical bone volume (BV), cross-sectional thickness (Cs.Th.), mean cross-sectional bone area (B.Ar), and mean cross-sectional tissue area (T.Ar) were all measured in two dimensions.A standard curve was established with a mineral density calibration phantom pair (0.25 g/cm3 CaHA and 0.75 g/cm3 CaHA) to determine mineral density.Intervertebral disc height and the length of the vertebral bones were measured and averaged along the dorsal, midline, and ventral regions in the sagittal plane.Disc height index (DHI) was calculated as previously described26,70 .

Figure 7 The
Figure 7

Figure 10 Opa1cKO
Figure 10 The quantitative data are represented as mean ± SEM or Box and whisker plots showing all data points with median and interquartile range and maximum and minimum values.Data distribution was checked with Shapiro-Wilk normality test, and differences between two groups were assessed by t-test or Mann-Whitney test as appropriate.One-way ANOVA, whereas non-normally distributed data were analyzed using or Kruskal Wallis test with appropriate post-hoc test (Sidak's multiple comparisons test) was used for comparisons between more than two groups.Analysis of Modi ed Thompson Grading data distribution and ber and ber thickness distribution were performed using chi-square test; p < 0.05.